Di-substituted 1,2-dioxetane compounds having increased water solubility and assay compositions

ABSTRACT

Stable, enzymatically triggered chemiluminescent 1,2-dioxetanes with improved water solubility are provided. Dioxetanes further substituted with two or more water-solubilizing groups disposed on the dioxetane structure provide superior performance by eliminating the problem of reagent carryover when used in assays performed on capsule chemistry analytical systems. Compositions comprising a dioxetane with two or more water-solubilizing groups, a non-polymeric cationic surfactant enhancer and optionally a fluorescer, for providing enhanced chemiluminescence are also provided.

This application is a divisional of application Ser. No. 08/509,305filed on Jul. 31, 1995 U.S. Pat. No. 5,777,135.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to stable 1,2-dioxetanes and compositionswhich can be triggered by chemical reagents, including enzymes, togenerate chemiluminescence.

The dioxetanes contain more than one ionizable group which are part ofan alkoxy substituent. Compositions used in the practice of theinvention contain a stable dioxetane as described above, a cationicsurfactant and optionally a fluorescer which enhance the amount ofchemiluminescence which is produced. Dioxetanes and enhancedcompositions of the present invention are useful in methods forgenerating light (chemiluminescence) and in methods of analysis fordetecting the presence or amount of an analyte. Importantly, theionizable groups afford a more water soluble dioxetane and solve anunexpected chemical carryover problem in capsule chemistry analyticalsystems.

(2) Description of Related Art

a. Enzymatically Triggerable Dioxetanes

The first examples of enzymatic triggering of dioxetanes are describedin a U.S. patent application (A. P. Schaap, U.S. patent application Ser.No. 887,139) and a series of papers (A. P. Schaap, R. S. Handley, and B.P. Giri, Tetrahedron Lett., 935 (1987); A. P. Schaap, M. D. Sandison,and R. S. Handley, Tetrahedron Lett., 1159 (1987) and A. P. Schaap,Photochem. Photobiol., 47S, 50S (1988)). The highly stableadamantyl-substituted dioxetanes bearing a protected aryloxidesubstituent are triggered to decompose with emission of light by theaction of both an enzyme and aqueous buffer to give a stronglyelectron-donating aryloxide anion which dramatically increases the rateof decomposition of the dioxetane. As a result, chemiluminescence isemitted at intensities several orders of magnitude above that resultingfrom slow thermal decomposition of the protected form of the dioxetane.U.S. Pat. No. 5,068,339 to Schaap discloses enzymatically triggerabledioxetanes with covalently linked fluorescer groups decomposition ofwhich results in enhanced chemiluminescence via energy transfer to thefluorescer. U.S. Pat. Nos. 5,112,960 and 5,220,005 and a PCT application(WO88/00695) to Bronstein disclose triggerable dioxetanes bearingsubstituted adamantyl groups. U.S. Pat. No. 4,952,707 to Edwardsdiscloses phosphate-substituted dioxetanes. A PCT application(WO94/26726) to Bronstein discloses adamantyl dioxetanes bearing aphenyl or naphthyl group substituted at a non-conjugated position withan enzyme labile OX group and with an additional group on the aryl ring.

Other triggerable dioxetanes are disclosed in a PCT application(WO94/10258) to Wang. The dioxetanes disclosed in Wang contain an alkoxygroup which may be mono-substituted and a substituted phenyl-OX groupwherein one or more non-hydrogen groups are present on the benzene ringsubstituent in addition to the triggerable OX group.

Dioxetanes disclosed in all of the foregoing publications generate alight-emitting carbonyl compound comprising an alkyl ester of anaromatic carboxylic acid, typically the methyl ester of a hydroxybenzoicor hydroxynaphthoic acid or else a hydroxyaryl ketone.

b. Surfactant Enhancement of Chemiluminescence from TriggerableDioxetanes

Enhancement of chemiluminescence from the enzyme-triggered decompositionof a stable 1,2-dioxetane in the presence of water-soluble substancesincluding an ammonium surfactant and a fluorescer has been reported (A.P. Schaap, H. Akhavan and L. J. Romano, Clin. Chem., 35(9), 1863(1989)). Fluorescent micelles consisting of cetyltrimethylammoniumbromide (CTAB) and 5-(N-tetradecanoyl)amino-fluorescein capture theintermediate hydroxy-substituted dioxetane and lead to a 400-foldincrease in the chemiluminescence quantum yield by virtue of anefficient transfer of energy from the anionic form of the excited stateester to the fluorescein compound within the hydrophobic environment ofthe micelle.

U.S. Pat. Nos. 4,959,182 and 5,004,565 to Schaap describe additionalexamples of enhancement of chemiluminescence from chemical and enzymatictriggering of stable dioxetanes in the presence of micelles formed bythe quaternary ammonium surfactant CTAB. Fluorescent micelles alsoenhance light emission from the base-triggered decomposition of hydroxy-and acetoxy-substituted dioxetanes.

U.S. Pat. No. 5,145,772 to Voyta discloses enhancement of enzymaticallygenerated chemiluminescence from 1,2-dioxetanes in the presence ofpolymers with pendant quaternary ammonium groups alone or admixed withfluorescein. Other substances reported to enhance chemiluminescenceinclude globular proteins such as bovine albumin and quaternary ammoniumsurfactants. Other cationic polymer compounds were marginally effectiveas chemiluminescence enhancers; nonionic polymeric compounds weregenerally ineffective and an anionic polymer significantly decreasedlight emission. A PCT application (WO 94/21821) to Bronstein describesthe use of mixtures of the aforementioned polymeric quaternary ammoniumsurfactant enhancers with enhancement additives.

The enhancement and catalysis of a non-triggerable dioxetane by pyraninein the presence of CTAB is described (Martin Josso, Ph.D. Thesis, WayneState University (1992), Diss. Abs. Int., Vol. 53, No. 12B, p. 6305).

U.S. Pat. No. 5,393,469 to Akhavan-Tafti discloses enhancement ofenzymatically generated chemiluminescence from 1,2-dioxetanes in thepresence of polymeric quaternary phosphonium salts optionallysubstituted with fluorescent energy acceptors.

European Patent Application Serial No. 94108100.2 discloses enhancementof enzymatically generated chemiluminescence from 1,2-dioxetanes in thepresence of dicationic phosphonium salts. No documents disclose thecombination of an anionic fluorescer and a dicationic enhancer forenhancing chemiluminescence from a triggerable dioxetane. No example ofenhancement of substituted dioxetanes of the type of the presentinvention has been reported.

c. Triggerable Dioxetanes with Improved Water Solubility

The enzymatically triggerable dioxetanes are now undergoing widespreaduse as substrates for marker enzymes in numerous applications includingimmunoassays, gene expression studies, Western blotting, Southernblotting, DNA sequencing and the identification of nucleic acid segmentsin infectious agents. Despite the growing use of these compounds, thereare limitations to there use in some assay methods. Triggerabledioxetanes which are more water-soluble are desirable. As shown in thestructures below, it is especially desirable that the hydroxy dioxetaneformed by the dephosphorylation of a phosphate dioxetane by alkalinephosphatase remain highly soluble in water and buffered solutions and incompositions containing chemiluminescence enhancing substances. Suchdioxetanes and compositions are of importance in certain solution assaymethods for detecting hydrolytic enzymes or conjugates of hydrolyticenzymes. ##STR1##

As further background of the present invention and as more fullyexplained in the examples below, it has been found that use ofconventional chemiluminescent dioxetane reagents in assays performed onautomated instrumentation based on the principles of capsule chemistryanalysis results in carryover of reagent from one fluid segment toanother, resulting in potentially inaccurate measurements, erroneousresults, and imprecision due to non-reproducibility. Capsule chemistryanalysis is described in U.S. Pat. No. 5,399,497, which is fullyincorporated by reference herein. It has been postulated that, amongother possible means for overcoming the carryover problem, improvedwater solubility of the hydroxy dioxetane, in particular, mighteliminate or minimize carryover of this luminescent reactionintermediate into adjacent fluid segments of a capsule chemistryanalysis system.

Dioxetane compounds in commercial use do not incorporate anysolubilizing groups which are appended to an alkoxy group. As such,these dioxetanes are unsuitable for use in assay methods requiring zerocarryover. A suggestion of incorporating a solubilizing group into adioxetane has been made (U.S. Pat. No. 5,220,005). A dioxetane with acarboxyl group substituted on an adamantyl substituent is claimed,however, the preparation of such a dioxetane is not described.Significantly, there is no disclosure of what effect the addition of acarboxyl group had, if any, on solubility and other properties of thedioxetane. There is no teaching in the art of how many solubilizinggroups are required or what particular advantage might be conferred. Useof solubilizing groups which interfere with the removal of theprotecting group which initiates light emission or which otherwiseinterfere with light production would be of no value. Solubilizinggroups which would be removed during the luminescent reaction likewisewould not be useful.

The present invention demonstrates surprisingly, that incorporation ofone solubilizing group is insufficient to eliminate the carryoverproblem associated with the hydroxy dioxetane produced bydephosphorylation of a phosphate dioxetane. Phosphate dioxetanes whosehydroxy dioxetane product remains highly water soluble are providedherein to solve this problem. Further, enhanced compositions containingsuch phosphate dioxetanes are provided which produce efficientchemiluminescence when reacted with a triggering agent in an assay.

OBJECTS

It is an object of the present invention to provide stable,enzymatically triggered 1,2-dioxetanes with improved solubility inaqueous solution to produce chemiluminescence by the action of anenzyme. It is a second object of the present invention to provide1,2-dioxetanes further substituted with two or more water-solubilizinggroups disposed on the dioxetane structure. It is a further object ofthe present invention to provide a composition comprising a dioxetanewith two or more water-solubilizing groups, a non-polymeric cationicenhancer and optionally a fluorescer, for providing enhancedchemiluminescence. It is a further object of the present invention toprovide dioxetanes and compositions which, when used in assays performedon capsule chemistry analytical systems, eliminate the problem ofreagent carryover.

IN THE DRAWINGS

FIG. 1 is a diagram of a capsule chemistry analysis system in whichcarryover was determined to be a problem.

FIG. 2 is a profile of adjacent segments in the capsule chemistryanalysis system showing the observed luminescence attributed tocarryover as more fully described in the Examples below.

FIG. 3 is a further profile of adjacent segments observed in theexperiments which are more fully described in the Examples below andwhich established that the carryover was not optical in nature.

FIG. 4 is a further profile of adjacent segments observed in theexperiments which are more fully described in the Examples below andwhich established that the carryover was in fact chemical in nature.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to triggerable dioxetanes with improvedwater solubility. Such triggerable dioxetanes eliminate or minimizecarryover of the luminescent hydroxy dioxetane into adjacent segments incapsule chemistry analytical systems. Carryover can result fromsolubilization, deposition or precipitation of light-emitting materialof low water solubility into the fluorocarbon oil which serves as theisolating fluid in capsule chemistry systems. Reagent carryover can leadto inaccurate measurements, erroneous results and imprecision due tonon-reproducibility.

The present invention relates to stable 1,2-dioxetanes which can betriggered by chemical reagents, including enzymes, to generatechemiluminescence. Stable dioxetanes useful in practicing the presentinvention may be of the formula: ##STR2## wherein R₁ is a hydrophilicorganic group comprising a substituted alkyl, heteroalkyl, alkenyl oralkynyl group containing 1 to 20 carbon atoms, at least two groups whichprovide increased solubility in aqueous solution and optionallycontaining one or more atoms selected from the group consisting ofoxygen, nitrogen, sulfur, phosphorus, halogen and alkali metals, whereinR₃ and R₄ are each selected from acyclic, cyclic and polycyclic organicgroups which can optionally be substituted with heteroatoms and whichprovide stability to the dioxetane, wherein R₂ is an aryl ring groupselected from phenyl and naphthyl groups which can include additionalsubstituents and wherein X is a protecting group which can be removed byan activating agent to form an unstable oxide intermediate dioxetanecompound which decomposes and releases electronic energy to form lightand two carbonyl-containing compounds, one of which is anoxyanion-substituted ester compound containing the solubility increasinggroups, according to the reaction: ##STR3##

In one embodiment, the group R₁ is a straight or branched chain C₁ toC₂₀ alkyl group substituted with at least two groups which provideincreased solubility in aqueous solution. Preferred solubilizing groupscomprise groups which are substantially ionized under the conditions ofuse and include without limitation carboxylate, sulfonate, sulfate,phosphate, phosphonate, nitrate, trialkylammonium, trialkylphosphonium,dialkylsulfonium and guanidinium groups. Carboxylate groups are mostpreferred.

In a preferred embodiment, the group R₁ is a straight or branched chainC₁ to C₈ alkyl group substituted with at least two carboxylic acid orcarboxylate salt groups including, for example, groups of the structure##STR4## wherein M is selected from hydrogen, an alkali metal ion or aquaternary ammonium or phosphonium ion. When M is H it is recognizedthat the respective dioxetane compound will preferably only be usedunder conditions of pH where the carboxylic acid functions are ionized,i.e. pH≧about 7. Preferably M is a sodium or potassium ion.

In another embodiment, the group R₁ is a straight or branched chainheteroalkyl group substituted with at least two carboxylic acid orcarboxylate salt groups including, for example, groups of the structure##STR5## wherein M is as defined above.

The groups R₃ and R₄ in another embodiment are combined together in acyclic or polycyclic alkyl group R₅ which is spiro-fused to thedioxetane ring, containing 6 to 30 carbon atoms which provides thermalstability and which can include additional non-hydrogen substituents.##STR6##

The group R₅ is more preferably a polycyclic group, preferably anadamantyl group or a substituted adamantyl group having one or moresubstituent groups R₆ selected from halogens, alkyl, substituted alkyl,alkoxy, substituted alkoxy, carbonyl, carboxyl, phenyl, substitutedphenyl, amino and alkylamino groups covalently bonded thereto. ##STR7##

In another preferred embodiment the group R₂ is a phenyl or naphthylgroup. It is especially preferred that R₂ is a phenyl group in which theOX group is oriented meta to the dioxetane ring group as shown below.The phenyl ring may contain additional ring substituents R₇independently selected from halogens, alkyl, substituted alkyl, alkoxy,substituted alkoxy, carbonyl, carboxyl, amino and alkylamino groups.Some exemplary structures include by way of illustration ##STR8##

The OX group may be selected from hydroxyl, O⁻ M⁺ wherein M is selectedfrom hydrogen, an alkali metal ion or a quaternary ammonium orphosphonium ion, OOCR₈ wherein R₈ is selected from the group consistingof an alkyl and aryl groups containing 2 to 8 carbon atoms andoptionally containing heteroatoms, OPO₃ ⁻² salt, OSO₃ ⁻ salt,β-D-galactosidoxy and β-D-glucuronidyloxy groups. When the OX group isan ionic group such as OPO₃ ⁻² salt or OSO₃ ⁻ salt, the water solubilityincreasing groups in R₁ must not also be this group. A particularlyeffective dioxetane compound for the chemiluminescent detection ofalkaline phosphatase in aqueous solution is dioxetane 1 below. Hydroxydioxetane 2 results from dephosphorylation of dioxetane 1. ##STR9## Forcomparison, dioxetane 3 which incorporates only one ionizable group wasprepared. This dioxetane did not eliminate the carryover problemdiscussed above. ##STR10##

In another aspect of the invention, compositions providing enhancedchemiluminescence are provided. Enhanced compositions are advantageousin assays requiring the highest analytical sensitivity. Increasing thechemiluminescence efficiency of the dioxetane decomposition reactionwhile maintaining or reducing extraneous light emission from spontaneousdioxetane decomposition is one manner in which sensitivity can beenhanced or improved.

The present invention, therefore, also relates to compositionscomprising a stable 1,2-dioxetane which can be triggered to generatechemiluminescence and an enhancer. Compositions for providing enhancedchemiluminescence comprise a dioxetane as described above in an aqueoussolution, and a non-polymeric cationic enhancer substance whichincreases the quantity of light produced by reacting the dioxetane withthe activating agent compared to the amount which is produced in theabsence of the enhancer. It is preferred that the enhancer substance isa dicationic surfactant of the formula:

    Y.sup.- R.sub.3 A.sup.+ CH.sub.2 -Link-CH.sub.2 A.sup.+ R.sub.3 Y.sup.-

wherein each of A is independently selected from P and N atoms andwherein Link is an organic linking group containing at least two carbonatoms selected from the group consisting of substituted andunsubstituted aryl, alkyl, alkenyl and alkynyl groups and wherein Linkmay contain heteroatoms and wherein R is selected from lower alkyl oraralkyl containing 1 to 20 carbon atoms and wherein Y is an anion. It isespecially preferred that the enhancer substance is a dicationicsurfactant having the formula:

    Cl.sup.- (n-C.sub.4 H.sub.9).sub.3 P.sup.+ CH.sub.2 -Link-CH.sub.2 P.sup.+ (n-C.sub.8 H.sub.17).sub.3 Cl.sup.-

and wherein link is phenylene.

Compositions of the present invention for providing enhancedchemiluminescence may optionally contain at least one fluorescer as asupplementary enhancer. Fluorescers useful are those compounds which arecapable of increasing the quantity of light produced through energytransfer. Anionic fluorescers are particularly effective it is believeddue to favorable electrostatic interactions with the cationic enhancer.Particularly preferred fluorescers include, without limitation, pyranineand fluorescein.

EXAMPLES Example 1

Preparation of Dioxetane 1

This dioxetane was prepared by the sequence of reactions describedbelow. The synthesis up to the intermediate alkene(3-hydroxyphenyl)-(2-iodoethoxy)methylene!tricyclo- 3.3.1.1³,7 !decanewas conducted essentially as described in U.S. Pat. Nos. 5,013,827 and5,068,339. ##STR11##

(a) Synthesis of 2-chloroethyl 3-hydroxybenzoate

A solution of 65 g of 3-hydroxybenzoic acid (0.47 mol) in 500 mL of2-chloroethanol and 3 mL of H₂ SO₄ was refluxed for 4 hours. The cooledsolution was diluted with 500 mL of water and extracted with ethylacetate (3×250 mL). The ethyl acetate was extracted twice with aqueousNaHCO₃ and then with water. The ethyl acetate solution was dried andevaporated under reduced pressure yielding 85 g of product as a thickoil; ¹ H NMR (CDCl₃) δ3.814 (t,2H, J=6 Hz), 4.569 (t,2H, J=6 Hz), 5.36(br s,1H), 7.06-7.67 (m,4H). ##STR12##

(b) Synthesis of 2-chloroethyl 3-t-butyldimethylsilyloxybenzoate

A solution of 10 g (50 mmol) the ester from step (a),t-butyldimethylsilyl chloride (8.25 g, 55 mmol) and imidazole (4.76 g,70 mmol) in 100 mL of DMF was stirred under argon for 1 h. The solutionwas poured into 100 mL of water and extracted with ether (3×50 mL). Thecombined ether solutions were extracted with 20 mL of water. The organiclayer was dried and evaporated giving 18 g of an oil which was purifiedby column chromatography using 0-2% ethyl acetate in hexane yielding14.4 g of the product as a colorless oil (91%); ¹ H NMR (CDCl₃) δ0.218(s,6H), 0.995 (s,9H), 3.81 (t,2H), 4.56 (t,2H), 7.05-7.65 (m,4H).##STR13##

(c) Synthesis of(2-chloroethoxy)-(3-t-butyldimethylsilyloxyphenyl)methylene!tricyclo3.3.1.1³,7 !decane

A three neck flask was purged with argon and charged with 400 mL ofanhydrous THF. Titanium trichloride (48 g, 0.3 mol) was added withstirring and the flask was cooled in an ice bath. Lithium aluminumhydride (6.0 g, 0.16 mol) was added in small portions causing a briefexothermic reaction. After all of the LAH was added, the cooling bathwas removed and the mixture warmed to room temperature. Triethylamine(30 mL) was added and the black mixture was refluxed for 1.5 hours underargon. A solution of adamantanone (14 g, 93 mmol) and 2-chloroethyl3-t-butyldimethylsilyloxybenzoate (10 g, 31 mmol) in 50 mL of dry THFwas added dropwise while reflux was continued. After an additional 10min, TLC (5% ethyl acetate in hexane) indicated conversion of the esterto new material so the mixture was cooled and diluted with 3 L ofhexane. The hexane was decanted, filtered through filter paper andevaporated leaving an oil which was purified by column chromatography onsilica gel, eluting with 0-3% ethyl acetate in hexane yielding 8.68 g ofalkene (65% based on ester); ¹ H NMR (CDCl₃) δ0.195 (s,6H), 0.983(s,9H), 1.78-1.98 (m,12H), 2.65 (br s,1H), 3.334 (br s,1H), 3.55 (t,2H),3.66 (t,2H), 6.85-7.29 (m,4H). ##STR14##

(d) Synthesis of (2-chloroethoxy)-(3-hydroxyphenyl)methylene!tricyclo3.3.1.1³,7 !decane

The silyl-protected alkene (8.0 g, 19 mmol) was dissolved in 100 mL ofdry THF and reacted with 5.2 g of tetrabutylammonium fluoride. Afterstirring 15 min, the solution was evaporated and the residue partitionedbetween 100 mL of ether and 100 mL of water. The water solution wasextracted with three 100 mL portions of ether. The combined organicsolutions were washed with three 100 mL portions of water, dried andevaporated. The residue (6.5 g) was chromatographed using 5-20% ethylacetate in hexane. This produced 4.78 g of oily product; ¹ H NMR (CDCl₃)δ1.78-1.98 (m,12H), 2.67 (br s,1H), 3.34 (br s,1H), 3.55 (t,2H), 3.69(t,2H), 4.91 (br s,1H), 6.77-7.19 (m,4H). ##STR15##

(e) Synthesis of (3-hydroxyphenyl)-(2-iodoethoxy)methylene!tricyclo3.3.1.1³,7 !decane

The chloroethoxy alkene (2 g) was dissolved in 30 mL of anhydrousacetone. Sodium iodide (9.3 g) was added and the solution refluxed for 4days. After cooling, the precipitate was filtered and washed with alittle CH₂ Cl₂. The washings and acetone were combined and evaporated.The residue was redissolved in CH₂ Cl₂ and washed with water and dried.The crude material was chromatographed using 25% ethyl acetate inhexane. The yield was 93% of a slightly yellow oil; ¹ H NMR (CDCl₃)δ1.78-1.98 (m, 12H), 2.65 (br s,1H), 3.19 (t,2H), 3.35 (br s,1H), 3.69(t,2H), 4.90 (br s,1H), 6.75-7.24 (m,4H). ##STR16##

(e) Synthesis of((3,3-biscarboethoxy)propoxy)-(3-hydroxyphenyl)methylene!tricyclo3.3.1.1³,7 !decane

Diethyl malonate (3.12 g) was dissolved in 25 mL of absolute ethanolcontaining 11.65 mL of a 21% solution of sodium ethoxide in ethanol. Thesolution was cooled in an ice bath and the iodoethoxy alkene (3.2 g) wasadded dropwise as an ethanol solution to the reaction mixture. Thereaction was refluxed over night. After cooling, the mixture wasevaporated and redissolved in ethyl acetate.

The ethyl acetate solution was extracted with water, dried andevaporated. The crude material was chromatographed using 15-25% ethylacetate in hexane. The yield of product was typically 42-48%; ¹ H NMR(CDCl₃) δ1.24 (t,6H), 1.78-1.97 (m, 12H), 2.11-2.17 (q,2H), 2.66 (brs,1H), 3.21 (br s,1H), 3.42 (t,2H), 3.63 (t,1H), 4.13-4.22 (m,4H), 5.00(br s,1H), 6.75-7.21 (m,4H). ##STR17##

(f) Synthesis of((3,3-biscarboethoxy)propoxy)-(3-(bis-(2-cyanoethyl)phosphoryloxy)phenyl)methylene!tricyclo3.3.1.1³,7 !decane

A flask containing 30 mL of CH₂ Cl₂ under a layer of argon was cooled inan ice bath. Pyridine (6.95 mL) was added followed by slow addition ofPOCl₃ (2.47 mL) and stirring continued for 15 min. A solution of thealkene from step (e) in 10 mL of CH₂ Cl₂ and 5 mL of pyridine was addeddropwise. The ice bath was removed and the solution stirred for 2 hours.To this solution was added 6.95 mL of pyridine and 6.1 g of2-cyanoethanol. The reaction mixture was stirred over night resulting information of a yellow precipitate. The mixture was added to 200 mL ofCH₂ Cl₂ and washed with 3×75 mL of water. The CH₂ Cl₂ extract was driedand evaporated. The crude product was purified by chromatography using70% ethyl acetate in hexane. ¹ H NMR (CDCl₃) δ1.25 (t,6H), 1.74-1.98 (m,12H), 2.10-2.17 (q,2H), 2.61 (br s,1H), 2.81 (t,4H), 3.21 (br s,1H),3.42 (t,2H), 3.59 (t,1H), 4.11-4.22 (m,4H), 4.39-4.46 (m,4H), 7.14-7.36(m,4H). ##STR18##

(g) Synthesis of(3,3-biscarboxypropoxy)-(3-phosphoryloxyphenyl)methylene!tricyclo3.3.1.1³,7 !decane, tetrasodium salt

The alkene (3.9 g) from step (f) was dissolved in 12 mL of acetone. Asolution of 1.04 g of sodium hydroxide in 3 mL of water was added. Thesolution was stirred for 19 hours during which time 3 mL of acetone wasadded to the flask. The liquid was decanted and the solid washed withmore acetone. After drying under vacuum, a white solid was obtained. ¹ HNMR (D₂ O) δ1.72-2.07 (m, 14H), 2.59 (br s,1H), 3.14-3.18 (m,2H), 3.40(t,2H), 7.01-7.34 (m,4H). ##STR19##

(h) Synthesis of4-(3,3-biscarboxy)propoxy)-4-(3-phosphoryloxyphenyl)!spiro1,2-dioxetane-3,2'-tricyclo 3.3.1.1³,7 !decane!, tetrasodium salt

The alkene (2.5 g) from step (g) was dissolved in 50 mL of D₂ O.Polymer-bound Rose Bengal (500 mg) was suspended in 50 mL of p-dioxaneand added to the water solution. The reaction mixture was cooled to5°-7° C., oxygen bubbling was started and the mixture irradiated with asodium lamp through a 5 mil sheet of KAPTON (DuPont). After a total of18 hours, the polymer beads were filtered off, the vessel was washedwith methanol and the combined solution concentrated to 25 mL. Theremaining solvent was removed by lyophilization.

Example 2

Preparation of Dioxetane 3

This dioxetane was prepared by the sequence of reactions describedbelow. The synthesis up to the intermediate alkene(3-carboxypropoxy)-(3-hydroxyphenyl)methylene!tricyclo- 3.3.1.1³,7!decane was conducted essentially as described in published EuropeanPatent Application No. 91113601.8. ##STR20##

(a) Synthesis of 3-chloropropyl 3-hydroxybenzoate

3-Chloro-1-propanol (161.6 g, 1.71 mol) was refluxed with3-hydroxy-benzoic acid (40.0 g, 0.29 mol) and a catalytic amount ofsulfuric acid for a total of 9 hours. The excess alcohol was removed byvacuum distillation. The resulting orange oil was diluted with 400 mL ofwater and neutralized to pH 7. The solution was extracted with ethylacetate (3×250 mL). The organic layer was washed with 100 mL of brineand dried over anhydrous sodium sulfate, filtered and concentrated underreduced pressure. The product was purified by column chromatography withto give 67.5 g of product which contained a small amount of the startingalcohol: ¹ H NMR (CDCl₃) δ2.24 (quint, 2H), 3.70 (t, 2H), 4.48 (t, 2H,J=6 Hz), 5.55 (s, 1H), 7.05-7.63 (m, 4H). ##STR21##

(b) Synthesis of 3-chloropropyl 3-(tert-butyldimethylsiloxy)benzoate

3-Chloropropyl 3-hydroxybenzoate (67.5 g) was dissolved in anhydrous DMF(100 mL) followed by and t-butyl-dimethylsilyl chloride (52.11 g). Thereaction mixture was stirred under Ar until the starting material wasconsumed. The reaction mixture was diluted with water (500 mL) andextracted with hexane (4×750 mL) and then with 2×250 mL of ethylacetate. The combined organic solutions were dried over sodium sulfate,concentrated under reduced pressure and partitioned a second timebetween 500 mL of water and hexane. Drying the organic solution andevaporating gave the silylated ester as white crystals (89.64 g, 94%). ¹H NMR (CDCl₃) δ0.219 (s, 6H), 0.998 (s, 9H), 2.24 (quint, 2H), 3.70 (t,2H), 4.470 (t, 2H), 7.03-7.64 (m, 4H). ##STR22##

(c) Synthesis of(3-tert-butyldimethylsilyloxylphenyl)-(3-chloropropoxy)methylene!tricyclo3.3.1.1³,7 !decane

Titanium trichloride (25.8 g, 0.167 mol) was added to dry THF (500 mL)in a dried 3 L three-necked flask under a head of Ar at 0° C. Lithiumaluminum hydride (3.01 g, 0.084 mol) was added in small portions withvigorous stirring. The reaction mixture was warmed to room temperatureand 23.3 mL of triethylamine was added dropwise. After the addition wascompleted, the reaction mixture was refluxed for 2 h. Heating wasstopped and a solution of 3-chloropropyl3-(tert-butyldimethylsilyloxy)benzoate (5.28 g, 0.016 mol) andadamantanone (7.23 g, 0.048 mol) in 100 mL of dry THF was added dropwiseto the refluxing mixture over a 45 min period. The reaction mixture wasstirred over night at room temperature. The black mixture was dilutedwith water and extracted with 3×300 mL of hexane. The combined organicsolutions were filtered, dried over sodium sulfate and concentratedunder reduced pressure. The residue was partially purified by flashchromatography (2% ethyl acetate/hexane) to give the product as aviscous oil which was taken on to the next step. 1H NMR (CDCl₃) δ0.200(S, 6H), 0.988 (S, 9H), 1.66-2.01 (m, 14H), 2.63 (br s, 1H), 3.23 (br s,1H), 3.538 (t, 2H, J=5.7 Hz), 3.640 (t, 2H, J=6.6 Hz), 6.75-7.22 (m,4H). ##STR23##

(d) Synthesis of (3-Chloropropoxy)-(3-hydroxyphenyl)methylene!tricyclo3.3.1.1³,7 !decane

The silyl-protected alkene (5.36 g slightly impure) was dissolved in 75mL of dry THF and placed under Ar. TBAF (4.16 g, 13.2 mmol) was addedand the reaction mixture stirred for 30 min at room temperature. Thesolvent was evaporated and the residue was dissolved in 100 mL of water.The solution was extracted with 3×125 mL of ether and the organic layerwas washed with brine and dried over Na₂ SO₄. Removal of solvent underreduced pressure and column chromatography with 10% ethyl acetate inhexane afforded 2.05 g of the deprotected alkene. ¹ H NMR (CDCl₃)δ1.78-2.01 (m, 14H) , 2.65 (br s, 1H), 3.22 (br s, 1H), 3.541 (t, 2H,J=6 Hz), 3.644 (t, 2H, J=6 Hz), 5.30 (S, 1H), 6.75-7.24 (m, 4 H).##STR24##

(e) Synthesis of (3-cyanopropoxy)-(3-hydroxyphenyl)methylene!tricyclo3.3.1.1³,7 !decane

Sodium cyanide (300 mg, 6.1 mmol) was added to a solution of thechloroalkene (815 mg, 2.4 mmol) in anhydrous DMSO (4 mL) forming apurple solution which was heated to 120° C. for 1 hr. The cooledsolution was diluted with ether (50 mL) and washed with water (3×25 mL).The ether layer was dried and concentrated under reduced pressure. Theproduct was obtained as an oil in 85% yield. ¹ H NMR (CDCl₃) δ1.77-1.97(m, 14H), 2.49 (t, 2H), 2.65 (br s, 1H), 3.19 (br s, 1H), 3.49 (t, 2H),5.04 (S, 1H), 6.75-7.24 (m, 4H). ##STR25##

(f) Synthesis of (3-carboxypropoxy)-(3-hydroxyphenyl)methylene!tricyclo3.3.1.1³,7 !decane

Sodium hydroxide (7 mL of 2N solution) was added to the nitrile (0.67 g,2 mmol) and the reaction mixture was refluxed for 36 h. The solution wascooled to room temperature and neutralized with acetic acid (1 eq.). Themixture was extracted with ethyl acetate. The organic layer was washedthree times with water, then brine and dried over sodium sulfate. Theproduct was concentrated under reduced pressure to an oil affording theacid (0.64 g, 91%). ¹ H NMR (CDCl₃) δ1.78-1.97 (m, 14H), 2.47 (t, 2H),2.65 (br s, 1H), 3.22 (br s, 1H), 3.45 (t, 2H), 5.83 (S, 1H), 6.74-7.22(m, 4H). ##STR26##

(g) Synthesis of(3-carbomethoxypropoxy)-(3-hydroxyphenyl)methylene!tricyclo 3.3.1.1³,7!decane

The carboxylic acid (660 mg, 1.9 mmol) from the previous step wasdissolved in 10 mL of CH₂ Cl₂. DCC (597 mg, 2.8 mmol), DMAP (23 mg) andmethanol (1 mL) were added and the solution stirred for 18 hours. Themixture was filtered to remove solid material and evaporated. The solidresidue was suspended in ether and filtered. The product was purified bycolumn chromatography with 30% ethyl acetate in hexane. ¹ H NMR (CDCl₃)δ1.77-1.96 (m, 14H), 2.42 (t, 2H), 2.65 (br s, 1H), 3.22 (br s, 1H),3.41 (t, 2H), 3.65 (s,3H), 5.15 (br s,1H), 6.74-7.22 (m, 4H). ##STR27##

(h) Synthesis of(3-carbomethoxypropoxy)-(3-(bis-(2-cyanoethyl)phosphoryloxy)phenyl)methylene!tricyclo3.3.1.1³,7 !decane

A flask containing 5 mL of CH₂ Cl₂ under a layer of argon was cooled inan ice bath. Pyridine (0.5 mL) was added followed by slow addition ofPOCl₃ (465 mg) and stirring continued for 15 min. A solution of thealkene (360 mg) from step (g) in 0.5 mL of 1:1 CH₂ Cl₂ /pyridine wasadded dropwise. The ice bath was removed and the solution stirred for135 min. To this solution was added 1.0 mL of pyridine and 0.69 mL of2-cyanoethanol. The reaction mixture was stirred for 4 hours resultingin formation of a white precipitate. TLC showed formation of twomaterials. Adding an additional 200 μL of cyanoethanol caused theprecipitate to dissolve but stirring over night was without effect. Thesolution was evaporated to dryness and the crude product purifiedchromatographically. ¹ H NMR (CDCl₃) δ1.79-1.98 (m, 14H), 2.41 (t,2H),2.61 (br s,1H), 2.80 (m,4H), 3.23 (br s,1H), 3.41 (t,2H), 3.65 (s,3H),4.32-4.48 (m,4H), 7.15-7.37 (m,4H). ##STR28##

(i) Synthesis of(3-carboxypropoxy)-(3-phosphoryloxyphenyl)methylene!tricyclo3.3.1.1.sup.3,7 !decane, trisodium salt

The alkene (142 mg) from step (h) was dissolved in 4 mL of acetone. Asolution of 36.4 mg of sodium hydroxide in <1 mL of water was added. Thesolution was stirred for 20 hours. causing formation of a precipitate.The liquid was decanted and the solid washed with more acetone followedby methanol. After drying under vacuum, 100 mg of a white solid wasobtained. ¹ H NMR (D₂ O) δ1.71-1.95 (m,14H), 2.23 (t,2H), 2.62 (brs,1H), 3.18 (br s,1H), 3.53 (t,2H), 7.04-7.36 (m,4H). ##STR29##

(j) Synthesis of 4-(3-carboxypropoxy)-4-(3-phosphoryloxyphenyl)!spiro1,2-dioxetane-3,2'-tricyclo 3.3.1.1³,7 !decane!

The alkene (26.8 mg) from step (i) was dissolved in 1.5 mL of D₂ O.Polymer-bound Rose Bengal (75 mg) was suspended in 1.5 mL ofp-dioxane-d₈ and added to the water solution. The reaction mixture wascooled to 5°-7° C., oxygen bubbling was started and the mixtureirradiated with a sodium lamp through a 0.005" sheet of KAPTON. After atotal of 30 min, ¹ H NMR indicated the reaction to be complete(disappearance of peak at δ2.6) so the polymer beads were filtered off.

Example 3

Discovery of Reagent Carryover Problem in Capsule Chemistry AnalysisSystem

The experiments described below were performed on a prototype capsulechemistry analysis system essentially as described by Kumar et al inU.S. Pat. No. 5,399,497, with the detection system configured to measurelight emission (luminescence). The method and apparatus comprisesfeeding a stream of fluid segments through a Teflon tube, where the tubehas an isolating layer of fluorocarbon oil on the inner surface. Sampleand reagents are aspirated into this tube, and the resulting liquidsegments are moved through the tube. Separation steps and washing stepswhich are required by heterogeneous immunoassay methods were facilitatedby means of magnets, which transferred magnetic particles from oneaqueous segment to another. The detection system was comprised of aphoton counter and a fiber optic read head, in which the fibers wereradially arranged around the Teflon tube to maximize the efficiency oflight collection.

The TECHNICON IMMUNO 1® TSH method (Bayer Corporation, Tarrytown, N.Y.,USA) was used as a representative immunoassay method for the testing ofluminogenic reagents. The method principle involved incubation of aspecimen containing the antigen TSH with a first reagent (R1), whichcontained a fluorescein-labeled antibody, and simultaneously with asecond reagent (R2), which contained an antibody-alkaline phosphatase(ALP) conjugate. Each antibody was specific for a different epitope onthe TSH antigen, so that formation of a "sandwich" was promoted betweenthese two antibodies and the TSH antigen. Magnetic particles containingbound anti-fluorescein were used to capture the sandwich, and theparticles were subsequently washed to remove unbound reagents. Theparticles were then exposed to the luminogenic reagent, which containeda substrate for ALP, and luminescence was measured.

The luminogenic R3 reagent was comprised of 0.2 mM CSPD (disodium3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo 3.3.1.1³,7!decan}-4-yl)phenyl phosphate, (Tropix, Inc., Bedford, Mass., USA), 3 mMpyranine (hydroxypyrenesulfonic acid), 1 mM MgCl₂, 1M diethanolaminebuffer (pH 10.0), 0.1% Triton X-100 and 0.1% NaN₃. The sequence ofevents on the capsule chemistry analysis system is depicted in FIG. 1 ofthe drawings. The fluid capsule or test package was comprised of sixliquid segments, each of which had a volume of 28 μl. Magnetic particles(1.4 μl of the magnetic particle reagent used in the TECHNICON IMMUNO 1system were aspirated into the first segment (MP), with the remainder offluid being particle wash buffer (25 mM Tris, pH 7.5, containing 0.2MNaCl, 0.1% Triton X-100 and preservative). R1 (10.4 μl of serum-basedsolution containing fluorescein-labeled antibody to TSH), R2 (10.4 μl ofserum-based solution containing antibody to TSH conjugated with ALP) andS (7.2 μl of serum sample) were aspirated into the second segment. Thenext two segments (W1 and W2) were comprised of the same wash bufferused above in the MP segment. The fifth segment was R3, of thecomposition described above, with the key elements being the luminogenicsubstrate and the luminescence enhancer. The sixth segment was aninter-test buffer (same as the particle buffer described above), whichwas used to isolate adjacent tests. Magnetic transfers are depicted bythe arrows in the FIG. 1. These transfers were facilitated by one of twomagnetic transfer assemblies (M1 or M2). After an incubation of 13minutes, during which sandwich formation occurred, M1 transferred themagnetic particles into the R1+R2+S segment to initiate capture. Afteran additional period of 6 minutes, M2 transferred the particles into thefirst wash segment. After an additional period of 12 seconds, M2transferred the particles into the second wash segment. After anotherperiod of 12 seconds, M2 transferred the particles into the R3 segment,and light emission from this segment was detected as the stream ofaqueous segments passed back and forth through the luminometer readhead.

Since the Teflon tube is transparent to light, a problem with lightpiping (or "optical carryover") was expected. Specifically, some of thephotons emitted from the R3 segment of an adjacent test could enter theTeflon material, propagate down the length of the tube and be scatteredinto the detector during the measurement of the signal of the test ofinterest. However, while a signal was detected in the adjacent tests, itdid not occur in the expected manner. Instead of declining rapidly withdistance from test N, peaks of light output were observed centeredaround the R3 segments of the adjacent test packages, as shown in FIG. 2of the drawings. In FIG. 2, test N produced a high level ofluminescence, approximately 7.5 million counts per seconds (cps). TestsN-1 and N-2 were aspirated into the tube before test N and preceded thistest through the luminometer, and tests N+1 and N+2 followed after testN. The analysis system recorded photons counted for each individual airand liquid segment in the stream. The profile in FIG. 2 represents theaverage of 10 replicate panels of 5 tests each corrected for backgroundluminescence signal produced in the absence of ALP. The reagent blankvalues subtracted from each data point were an average obtained from 10replicate panels of 5 tests each. The magnitude of the carryover signalwas computed by dividing the peak cps in each adjacent test by the peakcps in test N, expressed in parts per million (ppm). Another possibleexplanation for this behavior was physical carryover of ALP from test Ninto the neighboring tests in an unintended manner. This could happen,for example, if the tube contained particulate materials deposited onthe walls, which could disrupt the smooth motion of the liquid segmentsthrough the tube. However, placement of 10 mM inorganic phosphate in theR3 segments of the adjacent tests had no effect on the magnitude of thesignals in the adjacent tests. Since this amount of phosphate would haveinhibited ALP by at least 90% under these test conditions, thepossibility of physical carryover was ruled out.

To further rule out optical carryover, the fluorescent enhancer pyraninewas omitted from test N only, but present in the adjacent tests. As aresult, the magnitude of the signal in test N was lower by a factor ofapproximately 10. However, as shown in FIG. 3 of the drawings, theheight of the peaks in the adjacent tests did not change significantly.The fact that the carryover signal did not change in the adjacent testsproportionately clearly demonstrated that this carryover was notoptical.

An additional and unexpected type of carryover was the cause of thecarryover problem. It was found that the hydroxy dioxetane intermediatewas sufficiently soluble in the fluorocarbon oil used to coat the innerwall of the Teflon tube, such that the carryover was due to transfer ofdissolved hydroxy dioxetane intermediate via the oil into the R3segments of the neighboring tests. This process was tested by changingthe buffer of the R3 segments in the adjacent tests from 1M DEA at pH 10to 1M Tris at pH 7. At pH 7, dissolved hydroxy dioxetane intermediate inthese R3 segments is stable and does not emit light. As shown in FIG. 4of the drawings, this change in pH resulted in the complete eliminationof the side bands of luminescence. The residual minor carryover in theN+1 and N-1 tests was due to the anticipated optical carryover. Theseresults verified that the source of light emission in the peaks in theneighboring tests was "chemical carryover" of the hydroxy dioxetanederived from CSPD into the R3 segments of adjacent tests.

Example 4

Elimination of Observed Chemical Carryover with DicarboxylicAcid-Substituted Dioxetane 1

Table 1 shows the effect of using three other dioxetanes on the chemicalcarryover of the reaction intermediate. LUMIGEN PPD4-(methoxy)-4-(3-phosphoryl-oxyphenyl)!spiro1,2-dioxetane-3,2'-tricyclo3.3.1.1³,7 !-decane!, (Lumigen, Inc.,Southfield, Mich., USA), dioxetane 3, a monocarboxylic acid derivativeand dioxetane 1, a dicarboxylic acid derivative were each used in testformulations at the same concentration. The ppm column is the signal forthe N+1 test, which represents worst case behavior. The carryover of theunmodified parent compound, PPD, was found to be more than twice as highas that observed with CSPD. Surprisingly, the monocarboxylic acidderivative, dioxetane 3, showed a reduction of only 84% in the magnitudeof the chemical carryover. This indicated that a single charged groupwas insufficient to completely prevent solubilization of the reactionintermediate in the fluorocarbon oil. However, the dicarboxylic acidderivative was 100% effective, indicating that two charged groups werefully adequate to achieve the desired behavior.

                  TABLE 1                                                         ______________________________________                                        Reduction of Chemical Carryover                                               Compound         ppm    % Reduction                                           ______________________________________                                        LUMIGEN PPD      1640                                                         Dioxetane 3       260    84                                                   Dioxetane 1        0    100                                                   ______________________________________                                    

Example 5

The Role of Enhancers

As part of the optimization of a reagent based on dioxetane 1, a numberof enhancer materials was examined. At pH 9.6, enhancer A(1-trioctylphosphoniummethyl-4-tributylphosphoniummethylbenzenedichloride) increased the luminescent signal by a factor of 6.2, andenhancer B (poly(vinylbenzyltributylphosphonium chloride)) increased thesignal by a factor of 19.7. At pH 10.0, enhancer A increased the signalby a factor of 4.8, and enhancer B increased the signal by a factor of18.9.

Despite the fact that enhancer B achieved higher light intensities,enhancer A was preferred for use on the analysis system since it is alow molecular weight monomeric compound. Polymeric compounds, especiallyif they are polycationic, interact with serum components, causingprecipitation, which would pose significant problems for the operationof the analysis system.

Both fluorescein and pyranine were found to be effective assupplementary fluorescers in combination with enhancer A. Alone, thesefluorescers must be used at relatively high concentrations (3 mM) inorder to achieve an enhancement of about ten-fold. However, incombination with enhancer A, a synergistic effect was observed, in whicha comparable enhancement resulted at 100-fold lower concentrations offluorescer than needed in the absence of the enhancer. Tables 2 and 3show the extent of enhancement by pyranine and fluorescein,respectively, in the presence of 1 mg/ml of enhancer A.

                  TABLE 2                                                         ______________________________________                                        Enhancement by Pyranine with Enhancer A                                        Pyranine! (mM)                                                                              Enhancement Factor                                             ______________________________________                                        0.01           3.7                                                            0.02           7.3                                                            0.03           9.8                                                            0.04           12.2                                                           0.05           13.7                                                           ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Enhancement by Fluorescein with Enhancer A                                     Fluorescein! (mM)                                                                            Enhancement Factor                                            ______________________________________                                        0.01            2.6                                                           0.02            4.0                                                           0.05            7.1                                                           0.10            8.7                                                           ______________________________________                                    

Example 6

Optimized Formulation for Capsule Chemistry Analysis System

The above described observations have led to the development of anoptimized formulation for the capsule chemistry analysis system. Thisformulation is comprised of 0.1-1 mM dioxetane 1, 0-0.05 mM pyranine,0.1-5 mg/mL Enhancer A, 0-1 MM Mg⁺², 0.1-1M 2-amino-2-methyl-1-propanol(pH 10.0) and 0.01-1% Triton X-100. Use of this formulation results incomplete elimination of the chemical carryover problem and enhancedperformance.

The foregoing examples are illustrative only and not to be restrictive.The scope of the invention is indicated only by the appended claims.

What is claimed is:
 1. A composition for producing light comprising inan aqueous solution;(a) a stable dioxetane of the formula: ##STR30##wherein R₁ is a hydrophilic organic group comprising a substitutedalkyl, heteroalkyl, alkenyl or alkynyl group containing 1 to 20 carbonatoms and at least two groups which provide increased solubility inaqueous solution and optionally containing at least one oxygen atom,wherein R₃ and R₄ are each selected from the group consisting ofacyclic, cyclic and polycyclic organic groups which can optionally besubstituted with heteroatoms and which can optionally be joined togetherto form a cyclic or polycyclic ring group spiro-fused to the dioxetanering, wherein R₂ is an aryl ring group selected from the groupconsisting of phenyl and naphthyl groups which can include additionalsubstituents and wherein X is a protecting group which can be removed byan activating agent to produce the light; and (b) a non-polymericcationic enhancer substance which increases the quantity of lightproduced by reacting the dioxetane with the activating agent compared tothe amount which is produced in the absence of the enhancer.
 2. Thecomposition of claim 1 wherein the enhancer substance is a dicationicsurfactant of the formula:

    Y.sup.- R.sub.3 A.sup.+ CH.sub.2 -Link-CH.sub.2 A.sup.+ R.sub.3 Y.sup.-

wherein each of A is independently selected from the group consisting ofP and N atoms, wherein Link is an organic linking group containing atleast two carbon atoms selected from the group consisting of substitutedand unsubstituted aryl, alkyl, alkenyl and alkynyl groups and whereinLink can optionally contain heteroatoms and wherein R is selected fromlower alkyl or aralkyl containing 1 to 20 carbon atoms and wherein Y isan anion.
 3. The composition of claim 1 wherein the enhancer substanceis a dicationic surfactant having the formula:

    Cl.sup.- (n-C.sub.4 H.sub.9).sub.3 P.sup.+ CH.sub.2 -Link-CH.sub.2 P.sup.+ (n-C.sub.8 H.sub.17).sub.3 Cl.sup.-

and wherein link is phenylene.
 4. The composition of any one of claims1, 2 or 3 wherein the dioxetane has the formula: ##STR31## wherein R₅ isselected from the group consisting of cyclic and polycyclic alkyl groupswhich are spiro-fused to the dioxetane ring and which contain 6 to 30carbon atoms and which can optionally include additional substituents.5. The composition of claim 4 wherein R₅ is selected from the groupconsisting of an adamantyl group and a substituted adamantyl group. 6.The composition of claim 4 wherein the dioxetane has the formula:##STR32##
 7. The composition of claim 1 wherein the dioxetane has theformula: ##STR33## and the enhancer substance is1-trioctylphosphoniummethyl-4-tributylphosphoniummethylbenzenedichloride.